Determination of microcontaminants in sediments by on-line solid-phase extraction-gas chromatography-mass spectrometry.

Two simple and straightforward analytical procedures for the screening of sediment samples are reported. They involve extraction with ethyl acetate or methanol and subsequent analysis by means of gas chromatography-mass spectrometry (GC-MS) using large-volume injection (LVI) or solid-phase extraction (SPE). The latter, which was originally developed for the analysis of aqueous samples, can be used without any modification. In general, 10 ml of organic solvent were added to 2 g of sediment, and the mixture was shaken and allowed to stand overnight. The methanolic extracts were then diluted in water and subjected to preconcentration and analysis using on-line SPE-GC-MS. The ethyl acetate extracts were injected directly into the GC using LVI. Both methods were used for the detection and identification of microcontaminants during a monitoring study of the river Nitra (Slovak Republic). They included polyaromatic hydrocarbons (PAHs), chlorofluorohydrocarbons, alkoxylated and alkylated phenols and benzothiazole derivatives. Semi-quantitative profiles of the contaminants were constructed and provisionally interpreted. The results indicate that SPE-GC-MS, and also LVI-GC-MS, have good potential for a rapid screening of sediment samples and the identification of microcontaminants. The analytical procedures pose no problems, and the on-line set-up is user-friendly.

Characterisation of bacteria by matrix-assisted laser desorption/ionisation and electrospray mass spectrometry.

Chemical analysis for the characterisation of micro-organisms is rapidly evolving, after the recent advent of new ionisation methods in mass spectrometry (MS): electrospray (ES) and matrix-assisted laser desorption/ionisation (MALDI). These methods allow quick characterisation of micro-organisms, either directly or after minimum sample preparation. This review provides a brief introduction to ES and MALDI MS and a discussion of micro-organism characterisation capabilities. Some attention is devoted to the analysis of mixtures of proteins, lipids and other compounds, to the combination of polymerase chain reaction technology and MS, and to the analysis of whole bacteria and their lysates. The review of results produced hitherto is concluded with an outlook on future developments.

Characterisation of cholera toxin by liquid chromatography--electrospray mass spectrometry.

Cholera toxin, one of the toxins that may be generated by various strains of the bacterium Vibrio cholerae, can be considered as a substance possibly used in biological warfare. The possibilities of characterising the toxin by liquid chromatography electrospray mass spectrometry (LC-ES-MS) were investigated. The toxin can be detected by flow-injection (FIA) ES-MS of a dialysed solution and observation of the charge envelope signals of its A-unit and B-chain protein; sufficient information for identification by the molecular mass of either protein could be obtained for quantities in the order of 10 fmol. Confirmatory analysis was carried out by 2-mercaptoethanol reduction and FIA-ES-MS detection of the product proteins or by tryptic digest LC-ES-MS with ion chromatogram detection of most of the tryptic fragments of the A-unit and B-chain from the singly, doubly or triply charged ion signals. The confirmatory tryptic digest LC-ES-MS analysis could be achieved with quantities as low as 1 pmol. Possible biovariations in the toxin can mostly be determined by sequencing, where the amino acid composition of tryptic fragments of the A1-chain, T5 and T15, and of the B-chain, T1, T4 and T5, cover all known biovariations. Partial sequencing of cholera toxin, originating from a classical strain, O1/569B, was achieved by LC-ES-MS/MS of most tryptic fragments larger than three amino acid residues.

Simultaneous determination of eight lipid peroxidation degradation products in urine of rats treated with carbon tetrachloride using gas chromatography with electron-capture detection.

One of the major processes that occur as a result of radical-induced oxidative stress is lipid peroxidation (LPO). Degradation of lipid peroxides results in various products, including a variety of carbonyl compounds. In the present study eight different lipid degradation products, i.e., formaldehyde, acetaldehyde, acetone, propanal, butanal, pentanal, hexanal and malondialdehyde were identified and measured simultaneously and quantitatively in rat urine after derivatization with O-(2,3,4,5,6-pentafluorbenzyl)hydroxylamine hydrochloride, extraction with heptane and using gas chromatography-electron-capture detection (GC-ECD). The identity of the respective oximes in urine was confirmed by gas chromatography-negative ion chemical ionization mass spectrometry (GC-NCI-MS). Simultaneously measured standard curves were linear for all oxime-products and the detection limits were between 39.0 +/- 5.3 (n=9) and 500 +/- 23 (n=9) fmol per microl injected sample. Recoveries of all products from urine or water were 73.0 +/- 5.2% and higher. In urine of CCl4-treated rats an increase in all eight lipid degradation products in urine was found 24 h following exposure. ACON showed the most distinct increase, followed by PROPA, BUTA and MDA. It is concluded that the rapid, selective and sensitive analytical method based on GC-ECD presented here is well suited for routine measurement of eight different lipid degradation products. These products appear to be useful as non-invasive biomarkers for in vivo oxidative stress induced in rats by CCl4.

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and alpha-chlorohydrin in urine of rats treated with epichlorohydrin.

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [14C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and alpha-chlorohydrin (alpha-CH). The identity of CHPMA and alpha-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 micrograms/ml. The analysis of alpha-CH based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 micrograms/ml. CHPMA and alpha-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 micrograms/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and alpha-CH by rats treated with ECH were found to be 31 +/- 10 and 1.4 +/- 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For alpha-CH, the doe-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and alpha-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and alpha-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and alpha-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.

Comparative study of different thermospray interfaces with carbamate pesticides: Influence of the ion source geometry.

Sixteen carbamate pesticides that belong to four chemical classes (oxime-N-methylcarbamates, aryl N-methylcarbamates, N-phenylcarbamates, and methyl esters of substituted carbamic acids) were investigated via three different commercially available thermospray interfaces and ion sources that exhibit wide differences in source geometry. Comparisons were made between the three interfaces with respect to ion formation and sensitivity of detection. Experimental parameters were standardized to obtain comparable experimental conditions. Very similar mass spectra for most carbamates were obtained that illustrate independence from the geometry of the ionization and desolvation chambers of the interfaces. These findings are in sharp contrast to several literature reports. However, thermally labile carbamates gave unsatisfactory results with regard to spectral compatibility between the interfaces. Such differences were due to thermally assisted hydrolysis reactions that occur in the vaporizer probe prior to ionization and reflect differences in the vaporizer designs. The study proves conclusively that comparable spectra can be obtained under thermospray with different interfaces and mass spectrometers.

Chemical and glutathione conjugation-related degradation of fotemustine: formation and characterization of a glutathione conjugate of diethyl (1-isocyanatoethyl)phosphonate, a reactive metabolite of fotemustine.

Fotemustine is a chemotherapeutic drug for the treatment of melanoma. In this study, we investigated the metabolic and chemical stability of fotemustine with 31P-NMR and FAB-MS. In the absence of GSH, 95% of fotemustine decomposed rapidly into a reactive diethyl ethylphosphonate (DEP) isocyanate, both in rat liver S9 fraction and in HEPES buffer (pH = 7.4). DEP-isocyanate in turn hydrolyzed rapidly into diethyl (1-aminoethyl)phosphonate, which reacted subsequently with the parent DEP-isocyanate. The remaining 5% of fotemustine was shown to decompose via dechlorination into diethyl [1-(3-nitroso-2-oxoimidazolidin-1-yl)ethyl]-phosphonate. In the presence of GSH, hydrolysis of DEP-isocyanate was blocked, and a glutathione conjugate (DEP-SG) was formed instead. DEP-SG was relatively stable at 37 degrees C in HEPES buffer. Only two minor and as yet unidentified decomposition products were formed. Addition of N-acetyl-L-cysteine (NAC) to DEP-SG in HEPES buffer converted DEP-SG rapidly into the corresponding NAC conjugate of DEP-isocyanate (DEP-NAC). The formation of DEP-SG from DEP-isocyanate and GSH appeared to be spontaneous. The extent of formation of DEP-SG from fotemustine and GSH was equal in both enzymatically active and inactive rat liver S9 fractions. In the presence and in the absence of GSH, the half-lives of decomposition (t1/2) of fotemustine were 33 +/- 6 and 27 +/- 3 min, respectively. The formation of the DEP-isocyanate and 2-chloroethanediazohydroxide intermediates from fotemustine appeared to be rate limiting, and not the hydrolysis of the DEP-isocyanate nor its conjugation to GSH. Active or inactive rat liver S9 fractions accelerated the decomposition of fotemustine slightly; i.e., the t1/2 of fotemustine decreased from 39 +/- 3 to 29 +/- 1 min. Further knowledge of the metabolic and chemical stability of fotemustine and DEP-isocyanate will contribute to a better understanding of fotemustine-related cytostatic effects and toxic side effects and to the design of chemoprotection against undesired toxic side effects.

Ion formation of N-Methyl carbamate pesticides in thermospray mass spectrometry: The effects of additives to the liquid chromatographic eluent and of the vaporizer temperature.

The effects of three additives-ammonium acetate, ammonium formate, and nicotinic acid-to the liquid chromatographic (LC) eluent and of the vaporizer temperature on the ion formation of N-methyl carbamate pesticides in thermospray (TSP) mass spectrometry was investigated by using filament- or discharge-assisted ionization. Nineteen carbamates and 12 of their known environmental degradation products were used as model compounds. The additives cause a strong reduction in the abundance of the characteristic fragment ions [M + H - CH3NCO](+) and [M - H - CH3NCO](-) for some of the carbamates. The addition of nicotinic acid reduces the quasimolecular ion intensity and, in most cases, produces nicotinic acid adduct ions. The addition of ammonium acetate or ammonium formate increases the intensity of the quasimolecular ion and in most cases produces a base peak for the ammonium adduct ion. The combination of a suppression of fragmentation and an enhancement of quasimolecular ion formation produces an overall gain in sensitivity. As to more specific effects, the addition of the ammonium salts reduces the intensity of M(-•) with the chlorinated carbamate barban and suppresses the formation of "odd" adduct ions in the TSP mass spectra of most other carbamates. Monitoring the intensity of the fragment and the quasimolecular ion signal as a function of the probe stem temperature, and the related probe tip temperature, proved to be an easy method to study the thermal degradation of the carbamates. This monitoring procedure showed that methiocarb and its sulfone already suffer from thermal degradation at a stem temperature of 90°C and that these compounds will therefore present problems in quantitation with LC/TSP mass spectrometry.

On-line post-column Diels-Alder derivatization for the determination of vitamin D3 and its metabolites by liquid chromatography/thermospray mass spectrometry.

Liquid chromatography/thermospray mass spectrometry (LC/TSP MS) has been used for the determination of vitamin D3 and some of its metabolites, i.e. 1 alpha(OH) vitamin D3, 25(OH) vitamin D3, 1 alpha,25(OH)2 vitamin D3 and 24,25(OH)2 vitamin D3, using positive and negative ion detection. Using these two modes positional isomers can be identified. Detection in the negative ion mode was preferred because of the slightly higher sensitivity. The limits of detection, using multiple ion detection, are 50-100 nM (6-12 pmol injected). On-line post-column derivatization based on [4 + 2] cyclo-addition (Diels-Alder reaction) proceeds within 1 min at room temperature. If this step is included in LC/TSP MS, the detection limits of the analytes can be improved 7-70-fold depending on the analyte tested. The best results (detection limits down to 1 nM, i.e. 0.12 pmol injected) are obtained with discharge ionization in the negative ion mode.

Identification and quantitative determination of glutathione-related urinary metabolites of fotemustine, a new anti-cancer agent.

1. Potential sulphur-containing metabolites of the anticancer agent, fotemustine, were synthesized, namely thiodiacetic acid (TDA), S-2-hydroxyethyl N-acetyl-L-cysteine (2-HE-NAC), N-acetyl-L-cysteine (NAC), S-methyl N-acetyl-L-cysteine (M-NAC), S-carboxymethyl-L-cysteine (CM-Cys), S-carboxymethyl N-acetyl-L-cysteine (CM-NAC), their corresponding sulphoxides and sulphones. Their chemical structures and stabilities were confirmed and derivatization methods were developed for their analysis by sulphur-selective g.l.c. (g.l.c.-FPD) and g.l.c.-mass spectrometry. 2. Four methods for isolation of potential metabolites of fotemustine were developed. Quantification of metabolites, derived in various ways was carried out by g.l.c.-atomic emission detection (AED) or g.l.c.-mass spectrometry. 3. Male Wistar rats (n = 4) were given a single i.p. dose of 40 mg/kg fotemustine. Urine excretion of TDA (18.4 +/- 1.9% in 24 h) and TDA sulphoxide (12.0 +/- 1.6% in 24 h) was significant; 32.7 +/- 4.6% of the fotemustine dose was excreted as TDA, and TDA sulphoxide in 48 h. NAC was excreted in rat urine at 1% of the dose. No other potential glutathione-derived metabolites of fotemustine were excreted. 4. Male Wistar rats (n = 4) were also treated i.p. with fotemustine at 5, 20 and 40 mg/kg, to investigate dose dependency and the time course of excretion of TDA. Excretion of TDA in 48 h urine decreased from 32 +/- 2 to 17 +/- 2% dose (mean +/- SD) with increasing dose of fotemustine.

Determination of tetrahydrophtalimide and 2-thiothiazolidine-4-carboxylic acid, urinary metabolites of the fungicide captan, in rats and humans.

Capillary gas chromatographic (GC) methods using sulphur and mass selective detection for the qualitative and quantitative determination of tetrahydrophtalimide (THPI) and 2-thiothiazolidine-4-carboxylic acid (TTCA), urinary metabolites of the fungicide captan in rat and humans, were developed. Urinary detection limits were 2.7 micrograms/l for THPI and 110 micrograms/l for TTCA. Intraperitoneal and oral administration of captan to rats resulted in a 48-h cumulative urinary excretion of THPI of 1%-2% and 3%-9% of the dose, respectively. Cumulative urinary excretion of TTCA over 48 h ranged from 2% to 5% of the captan dose for the respective routes of administration. In urine of non-exposed human subjects, neither THPI nor TTCA could be detected. In urine of fruit-growers who were occupationally exposed to captan, both THPI and TTCA could be detected. Based on these results, THPI and TTCA are proposed as promising parameters for the biological monitoring of occupational exposure to captan.